Rational protein design. The main goal of modern protein chemistry is to be able to design proteins with desired functions. The approach taken by the majority of scientists has been called rational protein design, which is highly dependent on knowledge of protein structure in three dimensions and protein folding. While an extremely powerful tool, rational protein design is currently limited to a small subset of enzymes: those with well defined three dimensional structures. The classic examples include proteins such as subtilisin from several Bacillus sp. (Wells, Powers et al. Proc Natl Acad Sci USA. (1987). 84: 1219-1223.) and T4 lysozyme (Matsumura, Becktel et al. (1989). Proc Natl Acad Sci USA. 86: 6562-6566.). The three dimensional organization of the amino acid side chains of the protein must be known at high resolution, and often protein/substrate and mutant structures must be known as well. While this method is useful, it is expensive, time consuming and requires difficult predictions. Even with a complete three dimensional X-ray crystallography structure in hand, it has proven difficult to design proteins with specific desired activities. To address these problems, methods which allow the accelerated evolution of proteins in vivo or in vitro can take advantage of natural mutagenic mechanisms and their resulting variability.
Protein fusions. Protein (translational) fusion can be created by the joining of translational sequences from two different genes to create a hybrid protein molecule. These applications have traditionally included the study of gene expression in microorganisms and eukaryotes (Casadaban, Martinez-Arias et al. (1983). Recombinant DNA. Methods in Enzymology. 100: 293-308.).
The use of protein fusion in vitro to generate hybrid (chimeric) proteins has gained importance in the development of novel or multifunctional enzyme activities. Applications include using protein domains to aid in protein purification (Sherwood. (1991). TIBTECH. 9: 1-3.) or to tag proteins for delivery to specific cellular locations (Crozel, Lazdunski et al. (1984). FEBS Lett. 172: 183-188; Moore and Kelly. (1986). Nature. 321: 443-446; Roitsch and Lehle. (1991). Eur J Biochem. 195: 145-50.). Domain shuffling between different proteins shows promise to create protein products with unique uses, often to bind a particular enzymatic activity to a site of interest (Panayotatos, Fontaine et al. (1988). Molecular genetics of bacteria and phages: prokaryotic gene regulation. 174.), as a reporter system for protein-protein interactions (Fields and Song. (1989). Nature. 340: 245-246.), to study the relatedness of different proteins (Caramori, Albertini et al. (1991). Gene. 98: 37-44.) or as targeted pharmaceuticals (Pastan and FitzGerald. (1991). Science. 254: 1173-1177.). Finally, another promising application of protein fusion to biotechnology is in creating multi-catalytic enzymes which are important in biocatalysis since they represent an alternative to co-immobilization and chemical crosslinking to create multienzyme systems (Bulow and Mosbach. (1991). TIBTECH. 9: 226-231.).
Problems in the development of protein fusions. Unfortunately, the construction of functional hybrid proteins can require an extensive knowledge of a protein's structure and functional domains in order to select a proper site for fusion. Many attempts have failed to produce the desired properties (Bowie and Sauer. (1989). J Bio Chem. 264: 7596-7602; Ellis, Morgan et al. (1986). Proc Natl Acad Sci, USA. 83: 8137-8141; Guan and Rose. (1984). Cell. 37: 779-787; Hellebust, Murby et al. (1989). BioTech. 7: 165-168.). Random deletions can be made to fuse two domains, but this is typically done for only one domain at a time, and the cost and time involved in such trial and error efforts can be substantial. In addition, while some gene fusions can be used to stabilize proteins, unstable structures are often formed which are recognized by the cellular degrading machinery (Bowie and Sauer. (1989). J Bio Chem. 264: 7596-7602; Hellebust, Murby et al. (1989). BioTech. 7: 165-168.). Also, even with the advanced level of molecular biological techniques available today, cloning remains a labor-intensive procedure, the results of which are not trivially predictable.
Several tools have been developed to make the construction of protein fusions simpler. These tools include new plasmid systems with convenient restriction sites (Shapira, Chou et al. (1983). Gene. 25: 71-82.), and a method for making gene fusions using the Polymerase Chain Reaction (PCR) so that convenient restriction sites are not required (Horton, Hunt et al. (1989). Gene. 77: 61-8.). None of these approaches, however, offers a truly simple way of making random protein fusions which eliminates the labor-intensive, trial and error aspects of traditional techniques, especially in the case when at least one of the two domains being studied has not yet been well characterized.
Transposons. Transposable elements are mobile stretches of DNA which are defined by two end terminals, usually denoted attL and attR at the left and right attachment ends respectively. Natural transposable elements contain DNA coding for transposases, and often portable genes conferring traits such as resistance to antibiotics. Some transposable elements can insert randomly into targeted DNA while others are sequence specific in their insertion sites. Transposons have been implicated as having a major role in evolution, and there is evidence for natural multifunctional enzymes having originated from the natural fusion of different protein domains.
Scientists have taken advantage of transposons to transport reporter genes for use in studying gene expression. These include transcriptional (Type I) fusions and translational (Type II) fusions. Transcriptional fusions, unlike translational fusions, place a reporter gene under the control of another promoter, but do not translationally fuse two protein domains. Translational fusions have generally been made to link a reporter gene carried inside the transposon to the translational frame of the target gene so that the reporter gene is expressed under direct control of the transcription and translation signals of the target gene of interest to study gene regulation. This requires that an open reading frame extend through the end of the transposable element to join an internal reporter protein to external translational sequences. This usually results in complete inactivation of the target gene.
Mu and the Transposing Bacteriophage. Bacteriophage Mu represents a class of transposons known as transposable bacteriophage which both function as a virus and a transposon. Mu replicates itself by transposing at high frequency, but can also integrate randomly into its host's genome as a lysogen. Mu is a model system for other transposable bacteriophage which are generally highly homologous. These include the Pseudomonas phage D3112, D108, and several other phage.
Because of the randomness of Mu insertions, and the high levels of transposition which can be generated by Mu strains containing a temperature sensitive transposition repressor (Mucts strains), Mu has been developed into a genetic tool to study gene expression in bacterial systems. Transposition of Mu derivatives has allowed scientists to perturb and examine the basic components critical to protein expression and translation. The most commonly used Mu derivatives include reporter genes which have been integrated into the Mu genome.
Type I Fusions. Type I transcriptional fusions have been used to study gene expression and regulation by co-opting the native transcriptional signal to express the exogenous reporter gene. For example for gene expression in E. coli, yeast, and Drosophila development.
Type II Fusions. Type II fusions have also been used to study gene expression and regulation, but in this case not only co-opt the transcriptional signals, but any translational signals as well to express the reporter gene. In this type of system the protein product usually only expresses the activity of the reporter exogenous gene.
MudII elements are mini-Mu deletion elements which are type II Mu transposable elements. Examples of these include .beta.-galactosidase fusion elements, where a .beta.-galactosidase (lacZ) reporter gene is inserted via transposable elements to detect transcription and translation of regulated gene systems. This usually results in the inactivation of the targeted gene.
Two types of Mu protein fusions have been developed, lacZ fusion elements and nptI fusion elements (Symonds, Toussaint et al. (1987). Phage Mu) The lacZ elements have been used to study translation regulation, determination of the translation phase of target genes, infer the location of a protein fusion by hybrid protein size, determine amino terminal sequence, and raise antibodies to regions of the protein of interest. By far the major goal of these studies has been to determine mechanisms of gene expression in the studied organisms.
The nptI system was designed to perform transposon-tagging since nptI is known to function as an aminoglycoside resistance gene in a variety of organisms. Transposon tagging is a method of creating an mutant by inserting a transposon with a selectable marker into the gene of interest so that mutants which inactivate the gene can be identified and maintained. This element is useful since it allows the nptI to be directly linked to the transcription/translation system of the organism being studied.
In these studies there has been no emphasis on creating novel proteins with new activities using these transposable elements. More importantly, these Mu elements are restricted to making amino-terminal fusions to the reporter protein. In these cases the inserted reporter gene is fused to the carboxy-end of the truncated targeted protein, terminating inside the Mu. If the transposable element were to insert before the amino terminal of a targeted gene, functional translation could only occur on the marker gene by itself, and no translation of the target gene would occur.
Problems with Mu. Unfortunately, available Mu elements had several problems. First, it has not been demonstrated that Mu elements can be readily used as a general method for the development of fusion proteins with two active domains. Second, the Mu elements used thus far for creation of protein fusions can not be used for construction of "carboxy-terminal" fusions since they did not have an open reading frame extending into the element. Third, the Mu elements previously used have long linker regions which incorporate a 40 amino acid linker between the fused domains. This could create protein folding problems or unwanted domain interactions. Fourth the currently existing Mu elements had only a single restriction site for the insertion of protein domains. Finally, although Mu elements which had deleted ends existed, it was not known whether they would transpose well with additional sequences added in such close proximity to the right end and whether the intervening linker region which would join the two protein domains would interfere with the construction of active chimeric proteins.
Other transposons. Other transposons have been used in a similar manner as Mu to create lac fusions to study gene expression. These include Tn10 and Tn917 (Berg and Howe. (1989). Mobile DNA).
The Tn5 element has also been used to construct phoA fusions in vivo. Fusions with alkaline phosphatase (phoA) have also been used to probe the structure of membrane bound proteins (Lloyd and Kadner. (1990). J Bacteriol. 172: 1688-93.). In general, these transposons have been used to study the membrane topology structure of a particular gene and protein secretion. The resultant fusion proteins are also limited to amino-terminal fusion of the reporter PhoA reporter protein resulting in fusion at the carboxy end of the targeted gene.
In general, these types of fusions have been applied to the study of gene expression. These elements were constructed with truncated marker proteins that extend through the end of the transposon. Transposition of the element can create an in-frame fusion with a target gene, thereby activating expression. Mini-Mu elements are used because they transpose at high frequencies, insert randomly, and can be packaged along with a target plasmid and transduced to a new cell (Symonds, Toussaint et al. (1987). Phage Mu). Some of the more pertinent work that has been done in the area of transposable elements are detailed in the following.
Namgoong et al., (1994), teach that the Mu transposition reaction attachment sites attL and attR can promote the assembly of higher order complexes held together by non-covalent protein-DNA and protein-protein interactions. (Namgoong, Jayaram et al. (1994). J Mol Biol. 238: 514-527.)
Harel et al., (1990), teach that in Mu helper-mediated transposition packaging the left end contains an essential domain defined by nucleotides 1 to 54 of the left end (attL). At the right end (attR), they teach that the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increase transposition frequency about 15-fold. (Harel, Dupliessis et al. (1990). Arch Microbiol. 154: 67-72.)
Groenen and van de Putte (1986), teach that the Mu A protein binds weakly to sequences between nucleotides 1 to 30 on the right end (R1) and between nucleotides 110 and 135 on the left end (L2). Mutations in these weak A binding sites have a greater effect on transposition than mutations of corresponding base pairs in the stronger A binding sites, located adjacent to these weak A binding sites. (Groenen and van de Putte. (1986). J Mol Biol. 189: 597-602.)
Groenen and et al. (1985) teach the DNA sequences at the end of the genome of bacteriophage Mu that are essential for transposition. (Groenen, Timmers et al. (1985). Proc Natl Acad Sci, USA. 82: 2087-2091.)
Lloyd and Kadner teach the how to probe the topology of the uhpT sugar phosphate transporter using a Tn5phoA element. (Lloyd and Kadner. (1990). J Bacteriol. 172: 1688-93.)
Phage Mu (1987), Cold Spring Harbor Laboratory Press (Symonds, et al eds.) teaches general methods for handling and working with bacteriophage Mu as a transposon, and describes the various uses of mini-Mu elements including the construction of Mu transcriptional and translational fusions.
Silhavy and Beckwith (1985) teaches the various uses of lac fusions for the study of biological problems. (Silhavy and Beckwith. (1985). Microbiol Rev. 49: 398-418.)
Mobile DNA, (1989), American Society for Microbiology, Publishers. (Berg, Howe, eds) describes transposons.
Casadaban, et al. (1983) Methods in Enzymol, provides a good general review of .beta.-galactosidase gene fusions for the study of gene expression. (Casadaban, Martinez-Arias et al. (1983). Recombinant DNA. Methods in Enzymology. 100: 293-308.)